1. Field of the Invention
The present invention relates to the fields of microbiology and microbial genetics. More specifically, the invention relates to novel bacterial strains, methods and processes useful for the fermentative production of amino acids.
2. Related Art
Following the recognition that Corynebacteria were useful for the fermentative production of amino acids (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464–468 (1957)), the industrial production of L-lysine became an economically important industrial process. Commercial production of this essential amino acid is principally done utilizing the gram positive Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum (Kleemann, A., et. al., “Amino Acids,” in ULLMANN'S ENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, vol. A2, pp.57–97, Weinham: VCH-Verlagsgesellschaft (1985)). These organisms presently account for the approximately 250,000 tons of L-lysine produced annually.
The efficiency of commercial production of L-lysine may be increased by the isolation of mutant bacterial strains which produce larger amounts of L-lysine. Microorganisms employed in microbial process for amino acid production are divided into 4 classes: wild-type strain, auxotrophic mutant, regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al., in Nutritional Improvement of Food and Feed Proteins, M. Friedman, ed., (1978), pp. 649–661). Mutants of Corynebacterium and related organisms enable inexpensive production of amino acids from cheap carbon sources, e.g., mollasses, acetic acid and ethanol, by direct fermentation. In addition, the stereospecificity of the amino acids produced by fermentation (the L isomer) makes the process advantageous compared with synthetic processes.
Given the economic importance of L-lysine production by the fermentive process, the biochemical pathway for lysine synthesis has been intensively investigated, ostensibly for the purpose of increasing the total amount of L-lysine produced and decreasing production costs (recently reviewed by Sahm et al., Ann. N. Y. Acad. Sci. 782:25–39 (1996)). Entry into the lysine pathway begins with L-aspartate (see FIG. 1), which itself is produced by transamination of oxaloacetate. A special feature of C. glutamicum is its ability to convert the lysine intermediate piperidine 2,6-dicarboxylate to diaminopimelate by two different routes, i.e., by reactions involving succinylated intermediates or by the single reaction of diaminopimelate dehydrogenase. Overall, carbon flux into the pathway is regulated at two points: first, through feedback inhibition of aspartate kinase by the levels of both L-threonine and L-lysine; and second through the control of the level of dihydrodipicolinate synthase. Increased production of L-lysine may be therefore obtained in Corynebacteria by deregulating and increasing the activity of these two enzymes.
In addition to the biochemical pathway leading to L-lysine synthesis, recent evidence indicates that the transportation of L-lysine out of cells into the media is another factor to be considered in the development of lysine over-producing strains of C. glutamicum. Studies by Krämer and colleagues indicate that passive transport of lysine out of the cell, as the result of a leaky membrane, is not the sole explanation for lysine efflux; their data suggest a specific carrier with the following properties: (1) the transporter possesses a rather high Km value for lysine (20 mM); (2) the transporter is an OH− symport system (uptake systems are H+ antiport systems); and (3) the transporter is positively charged, and membrane potential stimulates secretion (S. Bröer and R. Krämer, Eur. J. Biochem. 202: 137–143 (1991).
Several fermentation processes utilizing various strains isolated for auxotrophic or resistance properties are known in the art for the production of L-lysine: U.S. Pat. No. 2,979,439 discloses mutants requiring homoserine (or methionine and threonine); U.S. Pat. No. 3,700,557 discloses mutants having a nutritional requirement for threonine, methionine, arginine, histidine, leucine, isoleucine, phenylalanine, cystine, or cysteine; U.S. Pat. No. 3,707,441 discloses a mutant having a resistance to a lysine analog; U.S. Pat. No. 3,687,810 discloses a mutant having both an ability to produce L-lysine and a resistance to bacitracin, penicillin G orpolymyxin; U.S. Pat. No. 3,708,395 discloses mutants having a nutritional requirement for homoserine, threonine, threonine and methionine, leucine, isoleucine or mixtures thereof and a resistance to lysine, threonine, isoleucine or analogs thereof; U.S. Pat. No. 3,825,472 discloses a mutant having a resistance to a lysine analog, U.S. Pat. No. 4,169,763 discloses mutant strains of Corynebacterium that produce L-lysine and are resistant to at least one of aspartic analogs and sulfa drugs; U.S. Pat. No. 5,846,790 discloses a mutant strain able to produce L-glutamic acid and L-lysine in the absence of any biotin action-surpressing agent; and U.S. Pat. No. 5,650,304 discloses a strain belonging to the genus Corynebacterium or Brevibacterium for the production of L-lysine that is resistant to 4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose 2,4-dideoxy-L-arabinose or a derivative thereof.
More recent developments in the area of L-lysine fermentive production in Corynebacteria involve the use of molecular biology techniques to augment lysine production. The following examples are provided as being exemplary of the art: U.S. Pat. Nos. 4,560,654 and 5,236,831 disclose an L-lysine producing mutant strain obtained by transforming a host Corynebacterium or Brevibacterium microorganism which is sensitive to S-(2-aminoethyl)-cysteine with a recombinant DNA molecule wherein a DNA fragment conferring resistance to S-(2-aminoethyl)-cysteine and lysine producing ability is inserted into a vector DNA; U.S. Pat. No. 5,766,925 discloses a mutant strain produced by integrating a gene coding for aspartokinase, originating from Coryneform bacteria, with desensitized feedback inhibition by L-lysine and L-threonine, into chromosomal DNA of a Coryneform bacterium harboring leaky type homoserine dehydrogenase or a Coryneform bacterium deficient in homoserine dehydrogenase gene.
Many process designed utilizing bacterial mutant strains are designed to weaken bacterial growth and hence to enhance the yield of amino acid production through supplementation with other nutrients. Usually, mutants designed to improve the percent yield of an amino acid from substrates such as glucose will also lose their ability for vigorous growth like their wild type strains. Besides resulting in an overall decrease in amino acid yield, these mutants also require more nutrients to support their growth, which can increase the cost in the production significantly.
Thus, there is a continuing need-in the art for the development of novel amino acid producing bacterial strains that enable maximized yields of a particular amino acid at a low cost of production. In view of these problems, an alternative method comprises special mutants and media that is employed to increase the productivity and to decrease the ingredient cost.